Treatment for non-alcoholic fatty liver diseases

ABSTRACT

Mixtures of vitamin E and polyunsaturated fatty acids (PUFAs) are provided as agents for the prevention, control and/or treatment of conditions associated with excessive fat accumulation in the liver which is not caused by alcohol abuse, including control and/or treatment of non-alcoholic steatosis in the liver—known as non-alcoholic fatty liver disease (NAFLD)—and/or non-alcoholic steatohepatitis (NASH) in a subject in need thereof.

This application is a divisional of commonly owned copending U.S.application Ser. No. 15/536,252, filed Jun. 15, 2017 (now abandoned)which is the U.S. national phase of International Application No.PCT/EP2015/079526, filed Dec. 14, 2015 which designated the U.S. andclaims priority to CH Patent Application No. 01939/14, filed Dec. 15,2014, the entire contents of each of which are hereby incorporated byreference.

The present invention relates to the use of mixtures of vitamin E andpolyunsaturated fatty acids (PUFAs) as agents for the prevention,control and/or treatment of conditions associated with excessive fataccumulation in the liver which is not caused by alcohol abuse. Thisincludes prevention, control and/or treatment of non-alcoholic steatosisin the liver—known as non-alcoholic fatty liver disease (NAFLD)—and/ornon-alcoholic steatohepatitis (NASH) in a subject in need thereof. Inparticular, the present invention relates to the use of such compoundscomprising vitamin E and PUFAs as active ingredients in the manufactureof medicaments for the prevention, control and/or treatment ofconditions related to NAFLD.

Although the pathophysiology of fatty liver has not yet been fullyclarified, a generally accepted mechanism is the “two-hit” theory (Dayand James, 1998 Gastroenterology 114:842-845). The first hit correspondsto the accumulation of free fatty acids (FFA) in the liver, which can berelated to obesity, or more generally to metabolic syndrome (includingdiabetes, hypertension and dyslipidemia). The second hit refers to theperoxidation of these fatty acids due to the oxidative stress producedby different factors (Angulo and Lindor, 2001 Gastroenterology120:1281-1285).

The final result of the first hit is an excessive FFA balance, fromoversupply and/or failure in lipid beta oxidation, leading to fatty acidaccumulation in the liver which gives rise to the first lesions(Charlton et al., 2002 Hepatology 35:898-904). These initial impactsmake the liver more vulnerable to aggressive factors of the second hit,which is mediated by oxidative stress and pro-inflammatory cytokines(TNF-α, TGF-β, IL-6, IL-8). FFAs increase the expression of cytochromeP450 2E1 (CYP 2E1), a microsomal enzyme which takes part in theβ-oxidation of several FFAs, causing the release of reactive oxygenmetabolites (Weltman et al., 1998 Hepatology 27:128-133). Also some FFAsare metabolized by peroxisomal β-oxidation, generating additionalreactive oxygen metabolites (hydrogen peroxide, hydroxyl radicals) (Raoand Reddy, 2001 Semin Liver Dis 21:43-55). An excess of these moleculesdepletes natural antioxidants such as glutathione and vitamin E in theliver, causing oxidative stress which results in lipid peroxidation(Neuschwander-Tetri and Caldwell, 2003 Hepatology 37:1202-1219).Consequently damage in the hepatocyte organelles and membranes occurs,leading to hepatocellular degeneration and ultimately necrosis(Garcia-Monzon et al., 2000 J Hepatol 33:716-724). Lipid peroxidation inmitochondria results in extra production of reactive oxygen metabolites,causing more oxidative stress (Solis Herruzo et al., 2006 Rev Esp EnfermDig 98:844-874). Under these circumstances nuclear factor κB (NF-κB)will be activated, which stimulates the synthesis of inflammatorymediators such as pro-inflammatory cytokines (TNF-α, TGF-β, IL-8)(Angulo, 2002 N Engl J Med 346:1221-1231). Besides this, final aldehydeby-products of lipid peroxidation, such as malondialdehyde (MDA), and4-hydroxynonenal show chemotactic properties and activatepro-inflammatory cytokines (TNF-α, TGF-β, IL-6, IL-8), and stimulatehepatic collagen-producing stellate cells (Pessayre, 2007 JGastroenterol Hepatol 22 (Suppl 1):S20-S27). Patients with NAFLD had anincreased expression of TNF-α in the liver (Crespo et al., 2001Hepatology 34(6): 1158-1163). The final result, a mixed lesion is knownas steatohepatitis, characterized by steatosis, inflammatoryinfiltration and fibrotic degeneration of the liver tissue, and finallyhepatocyte necrosis. Ongoing oxidative stress and lipid peroxidationinduce continuous collagen production which leads to fibrosis reachingthe stage of hepatic cirrhosis (Chitturi and Farrell, 2001 Hepatology36:403-409).

The liver-specific Kupffer cells seem to play an important role in thepathogenesis of fatty liver diseases such as, e.g. NASH. Kupffer cellsare liver-resident macrophages providing significant protection againstendotoxins and harmful exogenous particles from the portal vein. Thepathogenesis of NASH may encompass hyperendotoxemia (Creely et al., 2007Am J Physiol Endocrinol Metab. 292:E740-E747) as a consequence ofimpaired phagocytotic function of Kupffer cells (Loffreda et al., 1998FASEB J. 12:57-65). Damaged clearance of bacterial metabolites,endotoxins, lipopolysaccharides etc. might speed up the pathogenesis ofliver diseases. Activation of Kupffer cells leads to additional stressstimuli and may determine the fate of hepatocytes from survival towardapoptosis. In Kupffer cells overproduction of cytokines, e.g. TNF-α andIL-1β occurs. Simultaneously, Kupffer cells become more sensitive tothese molecules (Diehl, 2002 Am J Physiol Gastrointest Liver Physiol.282:G1-G5). Inflammatory mediators produced by activated Kupffer cellstrigger hepatic stellate cells to synthesize collagen which might resultin liver fibrosis and cirrhosis. Aggregates of hypertrophic Kupffercells can be observed in perivenular areas of the livers of NASHpatients compared with the diffuse distribution seen in case of simplesteatotic liver (Park et al., 2007 J Gastroenterol Hepatol. 22:491-497).

With regards to the inflammatory events in the liver, such as associatedwith NAFLD or NASH, the effect of vitamin E has been examined in varioushuman clinical studies (Pacana et al. Curr Opin Clin Nutr Metab Care2012; 15:641-648). Through the intake of vitamin E, liver enzymes couldbe improved with a decrease of the plasma cytokine level. With a dailydose of 800 IU/day liver histology in non-diabetic adults withbiopsy-proven NASH could be improved. However, the use of vitamin E isnot recommended in diabetic patients as treatment of NASH, NAFLD withoutliver biopsy, NASH cirrhosis or cryptogenic cirrhosis (Chalasani et al.Hepatology, vol. 55, no. 6, 2012).

Therefore, there is a need for new agents with hepatoprotective effectsbut with weak or no side effects. These agents could be used forprevention, control and/or treatment of fatty liver diseases which arenot caused by alcohol abuse.

In accordance with the present invention it has been found that certaincompounds can modulate the biosynthesis/overproduction of inflammatorymediators which are involved in NAFLD or related malfunction of theliver, said mediators include e.g. eicosanoids (prostaglandins,leukotrienes), cytokines, chemokines, nuclear factors and/or nitricoxide.

Surprisingly, it has been found out that combinations of vitamin E andPUFAs exhibit hepatoprotective effects and are able to synergisticallymodulate the biosynthesis/overproduction of some pro-inflammatorymediators such as, e.g., cytokines. Therefore, such compounds are usefulfor the prevention, control and/or treatment of conditions associatedwith excessive accumulation of fat in the liver, preferably forprevention, control and/or treatment of NAFLD.

Thus, the present invention relates to the use of a mixture comprisingvitamin E and PUFAs for the treatment, control and/or prevention ofhepatic inflammation and cell injury in the liver, more preferablyprevention, control and/or treatment of NAFLD and related diseases.Thus, in one aspect, the present invention relates to the use of vitaminE and PUFAs in the manufacture of a medicament for the prevention,control and/or treatment of conditions requiring modulation ofinflammatory responses in liver cells, in particular the treatment andprevention of NAFLD.

Mixtures according to the present invention comprising vitamin E andPUFAs may be used as nutraceutical compositions, i.e. as supplement todietary compositions, i.e., (fortified) food/feed or beverages, or ascompositions in dosage unit form such as pharmaceutical compositions,e.g., tablets, granules, pastes or effervescent formulations which mayfurther comprise pharmaceutically acceptable carriers, excipients ordiluents, including, but not limited to, lubricants, colorants, wettingagents, fillers, disintegrants and flavorants. The pastes may be filledinto hard or soft gelatine capsules.

As used herein, the term “vitamin E” includes both natural and syntheticmixtures of tocopherols, including α-tocopherol, β-tocopherol,γ-tocopherol and δ-tocopherol. Tocopherol, which is liquid at roomtemperature, is a group of methylated phenolic compounds of the generalformula (I),

wherein R1, R3 and R4 are independently from each other hydrogen ormethyl groups; and wherein each * represents an individual chiralcenter. With regards to the tocopherol isoforms, R1, R3 and R4 are asfollows: α-tocopherol (R1=R3=R4=CH3); β-tocopherol (R1=R4=CH3, R3=H);γ-tocopherol (R1=H, R3=R4=CH3); δ-tocopherol (R1=R3=H, R4=CH3).

It is preferred that at least one of the substituents R1 and R3 informula (I) is CH3, more preferably is the use of α-tocopherol and/orγ-tocopherol.

The tocopherols of formula (I) have chiral carbon centers which areindicated by the asterisk (*) in the formula. The configuration at thesechiral centers is defined to be either R or S, a concept which is knownto the person skilled in the art.

For a given definition of residues R1, R3 and R4, the respectivetocopherols according to formula (I) exist in 8 different isomers due tothese chiral centers (i.e. (2R,4′R,8′R)-, (2R,4'S,8′R)-, (2R,4′R,8'S)-,(2R,4'S,8'S)-, (2S,4′R,8′R)-, (2S,4'S,8′R)-, (2R,4′R,8'S)- and(2S,4'S,8'S)-tocopherol). As used herein, the tocopherols are eitherpresent in the form of mixture of said chiral isomers or isomericallypure. The (2RS, 4′RS, 8′RS) tocopherol is also known as(all-rac)-tocopherol.

A particularly useful form of vitamin E is α-tocopherol which is used ina mixture with PUFA.

PUFAs are classified according to the position of the double bonds inthe carbon chain of the molecule as n-9, n-6 or n-3 PUFAs. Examples ofn-6 PUFAs are linoleic acid (C18:2), arachidonic acid (C20:4),γ-linolenic acid (GLA, C18:13) and dihomo-γ-linolenic acid (DGLA,C20:3). Examples of n-3 PUFAs are α-linolenic acid (C18:13),eicosapentaenoic acid (EPA, C20:5), and docosahexaenoic acid (DHA,C22:6). Especially EPA and DHA have attracted interest of the foodindustry in recent years. The most available sources of these two fattyacids are fish and the marine oils extracted from them or microalgae.

As used herein, the term “PUFAs” or “PUFA” refers to a fatty acid havinga backbone comprising 16 or more carbon atoms, (for example, 16, 18, 20or 22 carbon atoms (C16, C18, C20, or C22, respectively), and two ormore carbon-carbon double bonds in the backbone. As used herein, a“long-chain PUFA” (LC-PUFA) refers to a fatty acid having a backbonecomprising 18 or more carbon atoms, and two or more carbon-carbon doublebonds in the backbone, for example, C18:3n-3 (alpha-linolenic acid orALA). When the notation CA:Bn-X is used for a methylene-interruptedPUFA, the “CA” is the number of carbons (for example C18, C20 or C22), Bis the number of double bonds and X is the position of the first doublebond counted from the methyl end of the fatty acid chain.

As used herein, PUFAs encompass the free acid forms thereof, as well assalts and esters thereof. As used herein, the term ester refers to thereplacement of the hydrogen in the carboxylic acid group of a PUFAmolecule with another substituent. Examples of common esters includemethyl, ethyl, trichloroethyl, propyl, butyl, pentyl, tert butyl,benzyl, nitrobenzyl, methoxybenzyl and benzhydryl. Other esters of PUFAsare described in US 2010-0130608 A1, which is incorporated herein byreference.

PUFAs for use with the present invention include omega-3, omega-6, andomega 9 polyunsaturated fatty acids, and oxylipins derived therefrom.Exemplary omega-3 PUFAs for use with the present invention include, butare not limited to, α-linolenic acid (C18:3n-3), C18:4n-4, ω-3eicosapentaenoic acid (20:5n-3) (eicosapentaenoic acid), ω-3docosapentaenoic acid (docosapentaenoic acid), ω-3 docosahexaenoic acid(22:6n-3), docosatetraenoic acid (22:4n-6), and combinations thereof.Exemplary omega-6 PUFAs for use with the present invention include, butare not limited to, γ linolenic acid, linoleic acid, conjugated linoleicacid, arachidonic acid (20:4n-6), ω-6 docosapentaenoic acid, andcombinations thereof. In some embodiments, a PUFA oil for use with thepresent invention is all-cis.

In some embodiments, the PUFA comprises DHA, also known by its chemicalname (all-Z)-4,7,10,13,16,19-docosahexaenoic acid, as well as any saltsor derivatives thereof. Thus, the term DHA encompasses DHA ethyl ester(DHA-EE) as well as DHA free fatty acids, phospholipids, other esters,monoglycerides, diglycerides, and triglycerides containing DHA. DHA isan ω-3 PUFA.

In further embodiments, the PUFA comprises EPA, known by its chemicalname (all-Z)-5,8,11,14,17-eicosapentaenoic acid, as well as any salts orderivatives thereof. Thus, the term EPA encompasses the free acid EPA aswell as EPA alkyl esters and triglycerides containing EPA. EPA is an ω-3PUFA.

In some embodiments, the PUFA oil that is used to make the thermallystable emulsion, is substantially free of one or more specific fattyacids. For example, a PUFA oil that contains DHA-EE can be substantiallyfree of EPA. On the other hand, a PUFA oil that contains EPA-EE can besubstantially free of DHA.

Commercially available PUFAs suitable for use with the present inventioninclude, but are not limited to, Martek DHA™ S Oil (Martek BiosciencesCorp., Columbia, Md.), Rosemary-Free Martek DHA™ S Oil (MartekBiosciences Corp., Columbia, Md.), Microalgae DHA™ Oil (MartekBiosciences Corp., Columbia, Md.), OMEGAPURE® oils (Omega Protein Corp.,Houston, Tex.), MARINOL® Oils (Lipid Nutrition, Wormerveer, NL), MEG-3oils and powders (Ocean Nutrition Corp., Dartmouth, Calif.), Evogel(Symrise AG, Holzminden, Del.), Marine Oil (Arista Industries, Wilton,Conn.), and OMEGASOURCE® oils (Source Food Technology, Inc., Raleigh,N.C.).

A particularly useful form of PUFA is ω-3 PUFA which is used in amixture with vitamin E, in particular α-tocopherol in accordance withthe present invention, i.e. for the treatment, control and/or preventionof conditions associated with excessive fat accumulation in the liverwhich is not caused by alcohol abuse.

PUFA's are preferably used in a concentration so that the dailyconsumption by a human adult (weighing about 70 kg) is in the range offrom 10 mg/day to 4000 mg/day, preferably from 200 mg/day to 600 mg/day,more preferably about 400 mg/day. A food or beverage suitably containsabout 5 mg to about 1000 mg of a PUFA per serving. If the nutraceuticalcomposition is a pharmaceutical formulation such formulation may containa PUFA in an amount from about 10 mg to about 1000 mg per dosage unit,e.g., per capsule or tablet, or from about 10 mg per daily dose to about4000 mg per daily dose of a liquid formulation.

Vitamin E or its derivative is preferably used in a concentration sothat the daily consumption by a human adult (weighing about 70 kg) is inthe range of from 5 mg/day to 2000 mg/day, preferably 15 to 50 IU/day,more preferably 30 IU/day. A food or beverage suitably contains about 2mg to about 500 mg of vitamin E per serving. If the nutraceuticalcomposition is a pharmaceutical formulation such formulation may containvitamin E in an amount from about 5 mg to about 1000 mg per dosage unit,e.g., per capsule or tablet, or from about 5 mg per daily dose to about2000 mg per daily dose of a liquid formulation.

The term “subject” as used herein includes, all higher animals whereininflammatory events are known. In particular, a subject is a mammal,including animals or humans.

As used herein, a “fatty liver” or “excessive fat accumulation”associated with NAFDL or NASH means that the liver contains more thanabout 5 to about 10 wt % of fat.

As stated above, the compounds according to the present invention havehepatoprotective properties and are useful for the prevention, controland/or treatment of conditions involved in NAFLD or related malfunctionof the liver. They can also be used as an adjunct to the treatment of avariety of diseases or disorders caused by excessive non-alcoholic fataccumulation in the liver via modulation of biosynthesis/overproductionof inflammatory mediators in the liver cells.

In a particular embodiment, the compounds of the present invention areused for the prevention, control and/or treatment of conditionsassociated with excessive fat accumulation in the liver which is notcaused by alcohol abuse, preferably prevention, control and/or treatmentof NAFLD and NASH.

Thus, the present invention is particularly directed to the use of acombination of vitamin E and PUFAs as defined above (in the manufactureof a medicament/composition) for the prevention, control and/ortreatment of conditions requiring modulation of inflammatory responseassociated with accumulation of fat in the liver which is not caused byconsumption/abuse of alcohol, especially of those conditions mentionedabove.

In a further embodiment, compounds/mixtures of the present invention maybe used in combination with other nutraceutical compositions ortherapeutic agents known to those skilled in the art for treatment,control and/or prevention of inflammatory disorders in the liver byadministration prior to, simultaneously with or following theadministration of the compound(s) as disclosed herein.

Depending on the mode of administration, compounds/mixtures according tothe present invention consist substantially of vitamin E and PUFA—i.e.being the main active ingredients—with furthermore addition of binders,fillers, carriers, excipients including water, glycerol, etc. known tothe skilled person.

According to the present invention, the ratio of vitamin E andpolyunsaturated fatty acids which is administered might be in the rangeof about 1:1 to about 1:5, such as e.g., 1:2, or in the range of about4:1 or about 5:1 to about 20:1, calculated as weight ratio.Particularly, the vitamin E is calculated as α-tocopherol. Useful ratiosmight be In an embodiment, the polyunsaturated fatty acid and vitamin Ecalculated as a weight ratio of 0.2:1, 0.4:1, 0.6:1, 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 25:1 or 30:1, whereinthe vitamin E is calculated as α-tocopherol.

In order to determine anti-inflammatory properties of compounds orcombinations thereof, appropriate cells or cell lines (i.e. whole blood,macrophages, leukocytes) will be activated with inflammatory stimuli invitro in the presence of the compounds. This leads to the secretion ofpro-inflammatory prostaglandin E₂ (i.e. the product ofcyclooxygenase-2), nitric oxide (synthesized by inducible nitric oxidesynthase) and various cytokines and interleukins. Due to theiranti-inflammatory effects, compounds will reduce the level of the twometabolites. Similarly, the expression of genes of inflammatory pathwayswill be monitored by quantitative PCR or by micro-array analysis.Anti-inflammatory compounds reduce their expression levels. Additiveand/or synergistic effects of compounds will be identified both at thelevel of specific inflammatory parameters and more generally in the geneexpression profile related to the cellular inflammatory response.

The anti-inflammatory effect of a combined therapy with vitamin E andPUFAs can be demonstrated in stimulated macrophages including Kupffercells by determining the inhibition of the synthesis of cytokines thatreflect the inflammatory response.

In order to induce an in vitro ‘inflammatory response’ related to NAFLD,Kupffer cells, i.e. liver-resident macrophages, are an ideal cell typeto study the hepatoprotective effects of a test substance, such asmixtures of vitamin E and PUFA according to the present invention.Alternatively, murine macrophages RAW264.7 or even monocyte/macrophagespresent in peripheral blood cells can be used appropriate surrogatemodels (Raptis et al. J Hepatology 60, 625-632, 2014). For in vitroexperiments, cells may be seeded into microtiter plates or 12-wellplates and stimulated with lipopolysaccharide (LPS) without or withgraded amounts of the test substances. Vehicle concentrations (i.e.DMSO) are kept constant. Culture supernatants may be harvested afterappropriate periods of time (e.g. 24 hours). Cytokines and interleukinscan be measured by appropriate ELISA-based multiplex assays. Thepercentage of inhibition of the mentioned inflammatory mediators presentin the liver-specific cells at a given concentration of the testsubstances (compared to maximal production by LPS-stimulated cells) iscalculated and the putative synergistic effect of the test substancecomputed (see below).

The following examples are illustrative only and are not intended tolimit the scope of the invention in any way. The contents of allreferences, patent applications, patents and published patentapplications, cited throughout this application are hereby incorporatedby reference.

EXAMPLES Example 1: Synergistic Effect of Vitamin E and PUFA inLeucocytes Experimental Procedures

Leukocytes are obtained from healthy donors. Mononuclear cells (MNC) arepurified by Ficoll-Isopaque gradient centrifugation. Cells (at 3-8×106cells/mL) are cultured in phenol-red free RPMI 1640, supplemented with0.25% FBS, 0.1 mM NEAA, 50 U/mL penicillin, 50 μg/mL streptomycin and5×10-5 M 2-mercaptoethanol. Cells are stimulated with LPS (100 ng/mL)and IFN-γ (20 U/mL) for 2-24 h. Multiparametric kits for determinationof cytokines and chemokines are purchased from BIO-RAD Laboratories(Hercules, Calif.) and used in the LiquiChip Workstation IS 200 (Qiagen,Hilden, Germany). The data are evaluated with the LiquiChip Analysersoftware (Qiagen).

The algorithm developed by Chou and Tatalay is used to calculatesynergistic effects (Chou, T-C. Et Tatalay, P. A simple Biol. Chem. 252,6438-6442, 1977; Chou, T-C. Et Tatalay, P. Analysis of combined drugeffects—A new look at a very old problem. Trends in Biological Sciences.November 1983, p 450-454, 1983). Interactions are quantified by theCombination Index (CI). Briefly, the % of inhibition of theconcentration of each single substance alone or the mixture of both aredetermined. The affected fraction (fa) (values between 0 and 1) andunaffected fraction (fu) (1−fa), respectively, is calculated. Formedian-effect plots, log (fa/fu) is plotted against log (D), where Drepresents the concentration of each single compound alone or themixture of both. Using CalcuSyn software (Biosoft, Ferguson, Mo.), whichis based upon the method by Chou Et Tatalay, a CI is computed for everyfraction affected: a CI <1 reflects synergistic inhibition of therespective inflammatory parameter; if CI=1 the substances have additiveinteractions; when CI >1 the interaction of substances reflectsantagonism. It has also been observed that substances can havesynergistic or antagonistic interactions at given concentrations orratios, respectively (see e.g. Pappa et al. Quantitative combinationeffects between sulforaphane and 3,3′-generalized equation for theanalysis of multiple inhibitions in Michaelis-Menten kinetic systems. J.diindolylmethane on proliferation of human colon cancer cells in vitro.Carcinogenesis 28, 1471-77, 2007).

Results:

Cells are stimulated with LPS, an pathogen-derived component thatinduces inflammatory response, which is reflected in the expression ofinflammatory genes and the secretion of cytokines, interleukins andchemokines (Table 1). LPS induces a massive increase of the secretion ofinflammatory mediators such as TNF-alpha. Concomitantly, inflammatorymediators are secreted. These parameters are modulated by pre-incubatingcells with α-tocopherol or ω-3 PUFA prior to the stimulation with LPS.For instance, the pro-inflammatory cytokine TNF-γ is reduced by 29% and64%, respectively (Table 1a). IL-1beta and CCL4/MIP-1 b are alsosignificantly reduced by the two substances (Tables 1 b, 1c). Thus, thesubstances reduces the extent of the inflammatory stress in cells thatrespond to inflammatory stimuli.

When α-tocopherol and ω-3 PUFA s are combined at differentconcentrations and ratios, significant synergistic effects are observedin the inhibition of the production of the pro-inflammatory cytokineTNF-alpha, but also of the pro-inflammatory interleukin IL-1beta, aswell as the chemokine CCL4 (see Tables 1a-c, right column). Thesynergistic effects are most prominent for TNF-alpha.

TABLE 1 Synergistic effects of α-tocopherol and ω-3 PUFA in theinflammatory response of leukocytes. AT = α-tocopherol; for more detailssee text. 1a: synergism between AT and ω-3 PUFA in inhibiting TNF-alphapg/mL ± SD % inhibition Treatment TNF-alpha (vs LPS) p (vs LPS) Combinedindex LPS 5290 ± 240 — — — AT 10 μM + LPS 5505 ± 177 −4 0.415 AT 50 μM +LPS 3755 ± 21  29 0.012 AT 200 μM + LPS-α 3870 ± 14  27 0.014 — DHA 10μM + LPS 3190 ± 14  60 0.007 — DHA 20 μM + LPS 3400 ± 523 64 0.043 — DHA50 μM + LPS 3105 ± 163 59 0.009 — (AT 10 + DHA 10) + LPS 1810 ± 269 340.005 0.04 (AT 10 + DHA 20) + LPS 2845 ± 516 54 0.026 0.06 (AT 10 + DHA50) + LPS 3025 ± 417 57 0.022 0.16 (AT 50 + DHA 10) + LPS 1800 ± 14  340.002 0.18 (AT 200 + DHA 10) + 1705 ± 431 32 0.009 0.70 LPS 1b:synergism between AT and ω-3 PUFA in inhibiting CCL4/MIP-1beta pg/mL ±SD % inhibition Combined Treatment CCL4 (vs LPS) p (vs LPS) index LPS99000 ± 11341 — — — AT 10 μM + LPS 91400 ± 5657 7 0.485 AT 50 μM + LPS83900 ± 3111 15 0.210 AT 200 μM + LPS-α 87950 ± 3165 11 0.315 — DHA 10μM + LPS 79995 ± 3465 32 0.151 — DHA 20 μM + LPS 67000 ± 7523 62 0.079 —DHA 50 μM + LPS 37600 ± 778  62 0.024 — (AT 10 + DHA 10) + LPS 61700 ±6930 38 0.058 0.44 (AT 50 + DHA 10) + LPS 67800 ± 283  32 0.062 0.56 (AT200 + DHA 10) + 75359 ± 495  24 0.098 0.77 LPS 1c: synergism between ATand ω-3 PUFA in inhibiting IL-1beta pg/mL ± SD % inhibition CombinedTreatment IL-1beta (vs LPS) p (vs LPS) index LPS 6310 ± 170 — — — AT 10μM + LPS 6770 ± 438 −7 0.301 AT 50 μM + LPS 5735 ± 134 9 0.064 AT 200μM + LPS-α 5430 ± 113 14 0.026 — DHA 10 μM + LPS 5405 ± 742 14 0.235 —DHA 20 μM + LPS  4865 ± 1633 23 0.339 — DHA 50 μM + LPS 1440 ± 71  770.001 — (AT 10 + DHA 10) + LPS 3520 ± 71  44 0.002 0.41 (AT 10 + DHA20) + LPS 3645 ± 884 42 0.053 0.83 (AT 10 + DHA 50) + LPS 796 ± 41 870.001 0.63 (AT 50 + DHA 10) + LPS 3570 ± 113 43 0.003 0.54 (AT 50 + DHA50) + LPS 1013 ± 95  83 0.001 0.77 (AT 200 + DHA 10) + 3455 ± 106 45 0.002 0.95 LPS (AT 200 + DHA 50) + 1080 ± 28  83  0.001 0.95 LPS

Example 2: Soft Gelatin Capsule

Soft gelatin capsules are prepared by conventional procedures providinga dose of vitamin E of 5 to 1000 mg (e.g. α-tocopherol), such as e.g. 50mg, and at least one compound selected from the group of PUFAs asdefined above of 10 to 1000 mg (e.g. DHA), such as e.g. 200 mg, whereinthe amounts are the recommended daily doses. Other ingredients to beadded: glycerol. Water, gelatine, vegetable oil.

Example 3: Hard Gelatin Capsule

Hard gelatin capsules are prepared by conventional procedures providinga dose of vitamin E of 5 to 1000 mg (e.g. α-tocopherol) and at least onecompound selected from the group of PUFAs as defined above of 10 to 1000mg (e.g. DHA), wherein the amounts are the recommended daily doses.Other ingredients to be added: fillers, such as, e.g., lactose orcellulose or cellulose derivatives q.s.; lubricant, such as, e.g.,magnesium stearate if necessary (0.5%).

Example 4: Tablet

Tablets are prepared by conventional procedures providing as activeingredient 5 to 1000 mg of vitamin E (e.g. α-tocopherol), such as e.g.20 mg, per tablet and at least one compound selected from the group ofPUFAs as defined above of 10 to 1000 mg (e.g. DHA), and as excipientsmicrocrystalline cellulose, silicone dioxide (SiO2), magnesium stearate,crospovidone NF (which is a disintegratent agent) ad 500 mg.

Example 5: Soft Drink

An orange juice drink colored with beta-Carotene 10% CWS and withvitamin E (e.g. α-tocopherol) and at least one compound selected fromthe group of PUFAs as defined above (e.g. DHA) may be prepared accordingto Table 2 and 3.

TABLE 2 Soft drink ingredients Sugar syrup 64°Brix 156.2 g Sodiumbenzoate 0.2 g Ascorbic acid, fine powder 0.2 g Citric acid 50% w/w 5.0g Pectin solution 2% w/w 10.0 g Vitamin E 2-500 mg PUFA 5-1000 mg Juicecompound (see Table 3) 30.0 g Water to 250.0 g

First, sodium benzoate is dissolved in water whilst stirring. Stirringis continued and sugar syrup, ascorbic acid, citric acid, pectinsolution and juice compound are added one after the other. Do not use ahigh speed mixer. The bottling syrup is diluted with (carbonated) waterto one liter of beverage.

TABLE 3 Ingredients Juice compound Orange juice concentrate 65°Brix483.3 g Lemon juice concentrate 45°Brix 173.3 g Oily orange flavor  5.0g Beta-carotene 10% CWS as 10% stock solution  10.0 g Deionized water328.4 g

For preparation of the juice compound, deionized water is first added tothe juice concentrates with gently stirring to allow the juiceconcentrates to hydrate. Then, the oily flavor and beta-carotene 10% CWSstock solution are added and pre-emulsified in arotor-stator-homogenizer. Homogenization is performed in a high-pressurehomogenizer at 200 bar.

Typical serving of a soft drink can be 240 ml, with an amount in VitaminE (e.g. α-tocopherol) which is about 5-100 mg/serving and an amount inPUFA (e.g. DHA) which is about 5-500 mg/serving

Example 6: Synergistic Effect of Vitamin E and PUFA in Kupffer Cells

The present study is extended to macrophages isolated from murine orhuman liver: Adherent cells (i.e. Kupffer cells) are obtained from livertissue. Isolation and cultivation is done according to the methoddescribed by Li et al., Immonological Letters 158, 52-56, 2014 oraccording to any method known in the art. These cells are treated withcompounds as described above (see Example 1). These cells will beactivated with an inflammatory stimulus and the effect of substances andcombination thereof on the reduction of the inflammatory response isdetermined. With this set-up, identical effects as those described forblood cell derived macrophages (see Ex. 1) are obtained.

1. A method of treating a patient having non-alcoholic steatohepatitisand/or non-alcoholic fatty liver disease which comprises administeringto a patent in need of treatment for non-alcoholic steatohepatitisand/or non-alcoholic fatty liver disease a treatment effective amount ofa composition comprising vitamin E and polyunsaturated fatty acid (PUFA)in a weight ratio of the vitamin E to the PUFA of about 1:1 to about5:1.
 2. The method according to claim 1, wherein the vitamin E isselected from the group consisting of α-tocopherol, β-tocopherol,γ-tocopherol, δ-tocopherol and mixtures thereof.
 3. The method accordingto claim 1, wherein the PUFA is selected from the group consisting ofω-3, ω-6, ω-9 polyunsaturated fatty acids.
 4. The method according toclaim 3, wherein the PUFA is selected from the group consisting of ω-3α-linolenic acid, ω-3 eicosapentaenoic acid, ω-3 docosapentaenoic acid,ω-3 docosahexaenoic acid, ω-3 docosatetraenoic acid, ω-6 γ linolenicacid, ω-6 linoleic acid, ω-6 conjugated linoleic acid, ω-6 arachidonicacid, ω-6 docosapentaenoic acid, or combinations thereof.
 5. The methodaccording to claim 1, wherein the composition is administered as a dailydose of about 5 to about 2000 mg vitamin E and about 10 mg to about 4000mg PUFA.
 6. The method according to claim 1 wherein the compositionconsists essentially of the vitamin E and the PUFA.
 7. The methodaccording to claim 1, wherein the composition comprises 10 to 50 mg ofthe vitamin E.
 8. The composition according to claim 7, wherein thevitamin E is calculated as α-tocopherol or γ-tocopherol.
 9. The methodaccording to claim 1, wherein the composition is administered in theform of a tablet, soft drink or gelatin capsule having hepatoprotectiveeffect.
 10. The method according to claim 9, wherein the compositionfurther comprises at least one ingredient selected from the groupconsisting of water, vegetable oils, gelatin, lubricants, and cellulose.